Enrichment of rare circulating tumor cells (CTCs) in blood is typically achieved using antibodies to epithelial cell adhesion molecule (EpCAM), with detection using cytokeratin (CK) antibodies. However, EpCAM and CK are not expressed in some tumors and can be downregulated during epithelial-to-mesenchymal transition. A micro-fluidic system, not limited to EpCAM or CK, was developed to use multiple antibodies for capture followed by detection using CEE-Enhanced (CE), a novel in situ staining method that fluorescently labels the capture antibodies bound to CTCs. Higher recovery of CTCs was demonstrated using antibody mixtures compared to anti-EpCAM. In addition, CK-positive breast cancer cells were found in 15 of 24 samples (63%; range 1–60 CTCs), while all samples contained additional CE-positive cells (range 1–41; median = 11; P = .02). Thus, antibody mixtures against a range of cell surface antigens enables capture of more CTCs than anti-EpCAM alone and CE staining enables the detection of CK-negative CTCs.
Background: Despite therapeutic advances, AML remains a disease in which the majority of patients relapse after attaining remission. The presence of bone marrow MRD is associated with impending AML relapse. Prediction and prevention of relapse could improve outcomes, but most current MRD tests either require bone marrow aspirate or detect individualized molecular genetic features present in a minority of AML. Furthermore, bone marrow MRD assessments are impractical if performed frequently enough to detect most early relapses. We developed and tested a novel microfluidic chip device (MCD) that can quantitate cell numbers using automated methods and explored its ability to detect low levels of peripheral blood leukemic cells with aberrant immunophenotypes. Methods: The MCD contains sinusoidal capture channels that were coated with antibodies with specificity towards one of the commonly expressed markers found on immature myeloid cells--CD33, CD34 and CD117 (capture antigens). Initial spiking experiments used fluorescently labeled leukemia cell lines HL60 (CD33+), KG1 (CD34+), Kasumi1 (CD117+) spiked into a 5000 cell/mL suspension. Cell suspensions were passed through MCDs coated with a capture antigen known to be expressed on the tested cell line. These experiments established an efficient capture using a flow rate of 1mL/sec. Then, AML patients with an aberrant immunophenotype were enrolled in a pilot study either at the start of induction chemotherapy or prior to allogeneic stem cell transplant (SCT). For patients receiving induction chemotherapy, whole blood samples were obtained monthly starting at the time of remission assessment, or monthly starting prior to SCT. Buffered whole blood was passed through MCDs coated with a capture antigen known to be expressed on patient myeloblasts. The captured cells were then released, eluted, centrifuged and plated on a glass slide. Plated cell pellets were then labeled with fluorescent antibodies targeting surface proteins known to be aberrantly (either by lineage infidelity or asynchronous expression) expressed on the patients' AML blasts. Automated fluorescence microscopy was used to identify and quantify captured cells with the known aberrant immunophenotype of the AML blasts. Descriptive statistics described serial cell counts in patients maintaining remission and relapsing patients. Results: Of 31 patients who have been enrolled in the study to date, 13 had at least 3 post-remission MCD analyses. Of these patients, 6 had either morphologic relapse or persistent/rising marrow MRD. In these patients, there was a trend towards higher initial aberrant immunophenotype cell counts, with mean initial count = 59 (95% CI 1, 108), compared to other patients with mean initial count = 15 (95% CI 5, 25). Of 5 patients who relapsed with MCD data within 1 month prior to relapse, the mean absolute rise prior to relapse above minimum MCD cell count was 54 (95% CI 2, 105), in comparison to non-relapsing patients with mean rise of 9 (95% CI 3, 15). From the initial 16 patients, 10 underwent induction therapy (the other 6 were enrolled prior to SCT). In these 10 patients there was a non-significant association between peripheral blood aberrant immunophenotype cells and remission status following induction. A total of 8 patients underwent allogeneic SCT. Two of these patients had known bone marrow MRD at the time of SCT and had a statistically significant greater number of aberrant immunophenotypic cells pre-SCT (48 and 60) compared to the 6 MRD negative patients (median = 12, range 9, 42). Conclusions: A novel MCD assay can reliably capture and detect low numbers of AML blasts from peripheral blood using immunofluorescent imaging and automated cell counts to quantify leukemia cells with aberrant immunophenotypes. Because this method uses peripheral blood, frequent sampling is feasible and of minimal risk to patients. An ongoing clinical trial will further explore the associations between MCD-based cell enumeration and clinical endpoints in AML patients that were suggested in the pilot phase of this study. Because the MCD releases trace populations of viable cells, additional experiments, such as primary cell culturing and single cell sequencing, are possible. Figure Disclosures Foster: Bellicum Pharmaceuticals, Inc: Research Funding; Daiichi Sankyo: Consultancy; MacroGenics: Research Funding; Celgene: Research Funding. Fedoriw:Alexion Pharmaceuticals: Consultancy, Speakers Bureau. Zeidner:Celgene: Consultancy, Honoraria, Research Funding; AsystBio Laboratories: Consultancy; Merck: Research Funding; Covance: Consultancy; Pfizer: Honoraria; Agios: Honoraria; Daiichi Sankyo: Honoraria; Tolero: Honoraria, Research Funding. Coombs:Covance: Consultancy; Octopharma: Honoraria; Medscape: Honoraria; Cowen & Co.: Consultancy; Loxo: Honoraria; H3 Biomedicine: Honoraria; Dedham Group: Consultancy; Pharmacyclics: Honoraria; Abbvie: Consultancy. Mirkin:BioFluidica: Employment. Zomorrodi:BioFluidica: Employment. Toughiri:BioFluidica: Employment. Bartakova:BioFluidica: Employment. Carson:BioFluidica: Employment. Muller:BioFluidica: Employment.
Introduction: The measurement of circulating tumor cells (CTCs) in blood generally requires either anti-EpCAM for capture, anti-cytokeratin (CK) for detection, or both. However, EpCAM and CK are absent in some tumor cells, and both may be down-regulated during neoplastic progression or during epithelial-to-mesenchymal transition (EMT). Here we show capture on a micro-fluidic system using single or multiple antibodies, and CTC detection with CEE-Enhanced (CE), a novel in situ staining method that fluorescently labels the capture antibodies bound to the CTCs. Methods: Buffy coat cells isolated from 8 mL of blood were pre-incubated with either anti-EpCAM alone or with a mixture of antibodies to epithelial and mesenchymal cell surface antigens. CTCs were captured on a micro-fluidic channel. Identification of CTCs was determined with anti-CK and with CE to detect cells with the capture antibodies bound to the cell surface antigens. All cells scored as positive for CK or CE were, by definition, CD45-negative and DAPI-positive. Results: Using anti-EpCAM alone for capture, significantly more CTCs were detected by CE staining than with anti-CK in breast and prostate cancers. This indicated that CK-negative CTCs were captured but not detected and that some EpCAM-positive CTCs were CK-negative. Results using capture antibody mixtures varied from sample to sample but gave up to 2-fold higher CK-positive cells than anti-EpCAM only, and as much as 4-fold higher CE-positive cells. Control blood from healthy donors was CK and CE-negative. All CK-positive cells co-stained with CE, as determined with different fluorescent labels. In a clinical study of stage IV breast cancer, CTCs were isolated with an antibody mixture and sequentially stained with CK and CE. Fifteen of 24 samples (63%) contained CK-positive cells (range 1-60 CTCs) while 24 of 24 samples (100%) contained additional CE-positive cells (range 1-41; median=11; Wilcoxon test, p=0.02). The modest correlation coefficient (r = 0.57, p=0.004) for the number of CE-positive and CK-positive cells in each sample suggests that one or more different phenotypes of CTCs were being detected. Amplified Her2 was detected by FISH in isolated CK-positive CTCs, and also in CK-negative, CE-positive CTCs from Her2-positive patients, indicating these were tumor cells. Conclusions: The CEE-Enhanced staining technology enables the detection of CK-negative CTCs. This novel detection method, based on the in situ labeling of antibodies used for capture, greatly expands single and multi-antibody approaches to the study of rare circulating cells. Specifically, this allows the exploration and study of circulating cells not expressing CK such as highly de-differentiated tumor cells, stem cells or those tumor cells undergoing EMT. The clinical significance of these CK-negative CTCs is under investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5172. doi:10.1158/1538-7445.AM2011-5172
Blood contains a wealth of diverse tumor biomarkers, including circulating tumor cells (CTCs), extracellular vesicles (EVs), and cell free DNA (cfDNA) allowing us to advance development of technologies to aid in detection and management of cancer and other related diseases. Biomarkers released from tumor cells harbor important signatures of disease including mutations, copy number variations, methylation changes, and/or chromosomal rearrangements that can be used as biomarkers to monitor disease progression or to tailor individualized treatment options in this era of personalized medicine. We describe the use of the Liquid Scan, an automated liquid biopsy platform to interrogate lung cancer patient blood for CTCs, cfDNA and EVs.For CTC diagnostics, whole blood from patient sample is run through Biofluidica’s customized chips coated with cancer specific antibodies. CTCs bound to the chips were eluted live and used for standard screening tests and molecular characterization of the tumor. We were able to capture CTCs from all stages (I, II, III, IV) of the lung cancer patient blood samples using EpCAM antibodies coated on the chip surface. CTCs detected from Stage I patients using minimal blood samples (1 ml) suggests Biofluidica’s microfluidic chip technology can detect pre-symptomatic disease and early detection of residual disease or relapse. The automated Liquid Scan is also used for capture and isolation of cfDNA and EVs with different processing protocols, microfluidic chips and reagents. Whole blood or plasma is applied to the chip that contains 1.4 million diamond shaped posts. The specific biomarker is captured and released from the chip for analysis. Here we demonstrate the ability of the platform to efficiently capture cfDNA and EVs from cell lines and plasma from patient samples with minimal background resulting in the ability to further analyze these biomarkers for genetic mutations. As a measure of tumor burden a comparison is made on the relative abundance of circulating tumor DNA (ctDNA) compared to background level of cfDNA originating from normal cells. Biofluidica’s Liquid Scan automated platform will help to provide excellent patient care that delivers high quality data suitable to assist in clinical decisions as per cancer stage, response to treatment and early detection. Biofluidica’s Liquid Scan can reduce expensive and painful biopsy procedures generally used in the current health care system. Citation Format: Rolf Muller, Sangeetha Purushotham, Paul Diaz, Alena Bartakova, Judy Muller-Cohn, Jerry Lu, Veronica Cheung, Roksolana Melnychuk, Jennifer Barber-Singh, Elizabeth Fabio, Hatim Husain, Malgorzata Witek, Maryam Zomorrodi, Mateusz Hupert, Steven Soper, Matt Jackson, Catherine Chen. Detection of CTCs from whole blood of lung cancer patients using the automated Liquid Scan [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3984.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.